Porous silica microspheres having a silanol enriched surface

ABSTRACT

Chromatographic materials comprising porous silica microspheres having silanol-enriched and completely silanized surfaces are disclosed. Processes for preparing the specified chromatographic material are also disclosed.

This application is a continuation of application Ser. No. 07/117,430filed Nov. 6, 1987 which in turn is a continuation of Ser. No. 798,332,filed Nov. 1, 1985, both now abandoned.

BACKGROUND OF THE INVENTION

1. Field of the Invention

This invention relates to porous silica microspheres which are useful aschromatographic material.

2. Background of the Art

This invention represents improvements in chromatographic materialcomprising porous silica microspheres. One improvement resides in anenriched concentration of silanol groups on the surface ofcrush-resistant microspheres. The higher level of surface silanol groupsallows improved silanizations which produce microspheres having enhancedchromatographic properties.

U.S. Pat. No. 3,782,075, issued to Kirkland, discloses an improvedpacking material for chromatographic columns. The packing materialcomprises a plurality of uniform-sized porous microspheres having anaverage diameter of about 0.5 to about 20 μm. The microspheres consistessentially of a plurality of uniform-sized colloidal particles, havinga refractory metal oxide surface arranged in an interconnectedthree-dimensional lattice. The colloidal particles occupy less than 50%of the volume of the microspheres with the remaining volume beingoccupied by interconnected pores having a uniform pore sizedistribution.

U.S. Pat. No. 3,857,924, issued to Halasz et al., discloses a processfor the production of spherical, porous silica particles. The processcomprises treating an alkali polysilicate solution having a silicacontent of from about 5 to 7.5 percent by weight batchwise with a cationexchange material to remove cations, and thereafter batchwise with ananion exchange material to remove mineral acids. The treated solution isemulsified and coagulated in a water-immiscible organic medium therebyforming the silica particles. The silica particles are disclosed ashaving surfaces covered with a certain amount of silanol groups and areused as supports in chromatography, in catalytic processes as catalysts,as carriers for catalytically active materials, and so on.

U.S. Pat. No. 4,131,542, issued to Bergna et al., discloses a processfor preparing a low-cost silica packing for chromatography. The processinvolves spray drying an aqueous silica sol containing from 5 to 60weight percent silica to form micrograins. These porous silicamicrograins are acid-washed and sintered to effect a 5 to 20% loss insurface area.

U.S. Pat. No. 4,477,492, issued to Bergna et al., discloses a processfor preparing superficially porous microparticles for use inchromatography and as catalysts or catalyst supports. The processcomprises spray-drying a specified well-mixed slurry of coremacroparticles, colloidal inorganic microparticles and a liquid. Theresulting product is dried and sintered to cause a 5%-30% decrease insurface area.

U.S. Pat. No. 4,010,242, issued to Iler et al., discloses oxidemicrospheres having a diameter in the 0.5 to 20 μm range. Themicrospheres are produced by forming a mixture of urea or melamine andformaldehyde in an aqueous sol containing colloidal oxide particles.Copolymerization of the organic constituents produces coacervation ofthe organic material into microparticles containing the organicmaterial. The organic constituent can be burned out to form a powder ofuniform-sized porous microparticles consisting of an interconnectedarray of inorganic colloidal particles separated by uniform-sized pores.

U.S. Pat. No. 4,105,426, issued to Iler et al., discloses a powder ofdiscrete, macroporous microspheroids, each having an average diameter inthe range of 2 to 50 μm. Each microspheroid is composed of a pluralityof large colloidal particles joined and cemented together at theirpoints of contact by 1 to 10% by weight of nonporous, amorphous silica.The microspheroids have a high degree of mechanical stability and asurface area between about 80 and 110% of that of the large colloidalparticles. A process for the manufacture of the powder is alsodisclosed.

It is known that porous silica microspheres silanized with a uniformcoating of organosilyl groups are efficient chromatographic material forseparating various types of organic molecules from mixtures. In order tocovalently attach these silyl groups, there must be silanol (Si-OH)groups on the silica surface. Another important characteristic for achromatographic material is crush resistance so that beds of materialare stable for use at high pressure. It is known to strengthen poroussilica microspheres by heating at about 900° C. After heatstrengthening, there are very few silanol groups left on the surface ofthe silica. Instead, the surface is largely dehydroxylated to siloxanegroups (SiOSi) which generally do not react with silanizing agents. Achromatographic material comprising crush-resistant silica microspheresof uniform pore size distribution having a high surface concentration ofsilanol groups is desirable.

SUMMARY OF THE INVENTION

The present invention provides a chromatographic material comprisingimproved porous silica microspheres having an average diameter of about0.5 to about 35 μm. Substantially all of the microspheres have adiameter ranging from about 0.5 to about 1.5 times the average diameter.The microspheres consists essentially of a plurality of substantiallyuniform-sized colloidal particles, having a silica surface, arranged inan interconnected three-dimensional lattice. The colloidal particlesoccupy less than about 50 volume percent of the microspheres. Theremaining volume is occupied by interconnected pores having asubstantially uniform pore size distribution. The microspheres have atotal concentration of silanol groups of from about 6 to about 16μmol/m². In a preferred embodiment, the microspheres are preparedaccording to a process comprising contacting heat strengthenedthermally-dehyroxylated porous silica microspheres having a totalconcentration of silanol groups of less than about 5.5 μmol/m² withwater in the presence of HF or at least one basic activator selectedfrom the group consisting of quaternary ammonium hydroxides, ammoniumhydroxide, and organic amines at a temperature of from about ambienttemperature to about 100° C. for sufficient time to generate the desiredconcentration of silanol groups.

The invention also provides porous silica microspheres having thespecified physical and chemical properties and a completely silanizedsurface.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows separation of alkylbenzene and polarity test mixtures onporous silica micropheres having a completely silanized surface.

FIG. 2 shows degradation of porous silica microspheres having acompletely silanized surface.

FIG. 3 shows separation of a polarity test mixture on porous silicamicrospheres having a completely silanized surface.

FIG. 4 shows separation of a polarity test mixture on wide-pore poroussilica microspheres having a completely silanized surface.

FIG. 5 shows separation of a peptide test mixture containing mellitin onporous silica microspheres having a completely silanized surface.

FIG. 6 shows pressure-strength results for specified porous silicas.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a chromatographic material comprisingcrush-resistant porous silica microspheres having a silanol-enrichedsurface which has favorable sorptive properties for separating organiccompounds, especially basic compounds. In addition, these silicamicrospheres permit the preparation of silanized surfaces with enhancedchemical stability with regard to hydrolysis. The invention alsoprovides a chromatographic material comprising crush-resistant poroussilica microspheres having a completely silanized surface which areparticularly useful for separating basic organic compounds encountered,for example, in biochemical research.

As used herein, the expression chromatographic material means granulescapable of forming a packed bed or column having 1) sorptively activesurfaces or 2) surfaces capable of being coated with a sorptively activesubstance to form sorptively active surfaces. A mixture is passedthrough the bed or column and repeated interactions associated with thechemical nature of components of the mixture and the active surfaces ofthe chromatographic material cause a separation of the components. Theexpression "total concentration of silanol groups" refers to the numberof moles of silanol groups which are detectable by thermogravimetricanalysis (TGA) divided by the surface area of the silica microspheres(i.e. moles silanol groups per m²). It is known that the surface of thesilica microspheres can have a maximum concentration of exposed silanolgroups of about 8 μmol/m². Silanol groups in excess of this maximumconcentration are "buried" beneath the surface of the silica. TGA iscapable of measuring the sum of exposed surface silanol groups and"buried" silanol groups.

The expression "completely silanized surface" means that the surface ofthe silica has reached complete equilibrium with organosilyl groups. Inthis state, the organosilyl groups are tightly packed and form an"umbrella" over unreacted silanol groups. The maximum number oforganosilyl groups that can be attached to the surface of silica islimited by steric properties of selected organosilyl groups. It is knownthat a completely silanized surface of silica with a relatively openstructure (e.g. fumed or pyrogenic silica) has a maximum surfaceconcentration of trimethylsilyl groups of from about 4.5 to about 4.7μmol/m². In this case, about 60% of the total silanol groups areavailable for silanization before steric factors limit the reaction.Larger (bulkier) groups on a completely silanized surface are in lowerconcentrations. For example, a completely silanized surface oftriphenylsilyl groups has a maximum surface concentration of about 1.9μmol/m². Similarly, on compact forms of porous silica, such as themicrospheres of the present invention, all of the silanol groups on thesilica surface are not available to silanization. For example, acompletely silanized surface on porous silica microspheres having asurface area of 440 m² /g and an average pore diameter of 70-80Angstroms has about 4.0 to about 4.3 μmol/m² of trimethylsilyl groups.

The chromatographic material of the present invention comprises poroussilica microspheres which have an average diameter of from about 0.5 toabout 35 μm, preferably from about 0.5 to about 20 μm and mostpreferably from about 1.0 to about 10 μm. As used herein, the expression"average diameter" means the statistical average of the sphericaldiameters of the microspheres. The microspheres are substantiallyuniform in size which means that less than 5% of the microspheres have adiameter less than about 0.5 times the average diameter and less than 5%have a diameter greater than 1.5 times the average diameter. Preferably,the range is about 0.8 to about 1.2 times the average diameter.Furthermore, the microspheres have controlled pore dimensions and arelatively large pore volume.

The porous silica microspheres consist essentially of a plurality ofsubstantially uniform-sized colloidal particles. The particles have asilica surface and are arranged in an interconnected three-dimensionallattice that occupies less than about 50 volume percent of themicrospheres. The remainder of the microspheres is comprised ofsubstantially uniform-sized pores. The size of the pores contained inthe microspheres will depend on the size of the colloidal particles.

The average diameter of the pores in the microspheres of the presentinvention, at a pore diameter of about 1,000 Angstroms, is about halfthe calculated diameter of the ultimate spherical particles making upthe microspheres. This diameter is calculated from the followingequation:

    D=6000/DA

where D is the calculated diameter of the ultimate particle, d is thedensity of the solid inorganic material (e.g., 2.2 grams per cm³ foramorphous SiO₂) and A is the specific surface area of the microspheres,determined by nitrogen adsorption, as disclosed in Nelson et al.,Analytical Chemistry, 30: 1387 (1958). At about 100 Angstroms, the porediameter is about equal to the colloidal ultimate particle diameters andat about 50 Angstroms it is about one and a half times the colloidalparticle diameter.

Porous silica microspheres of the present invention have a totalconcentration of silanol groups of from about 6 to about 16 μmol/m²,preferably from about 8 to about 16 μmol/m². These microspheres can beprepared by contacting heat-strengthened thermally-dehydroxylated poroussilica microspheres having a total concentration of silanol groups ofless than about 5.5 μmol/m² with water in the presence of HF or a basicactivator. The silanol-enriched microspheres provide a chromatographicmaterial which exhibits high hydrolytic stability and a low adsorptionof basic compounds. The silanol-enriched microspheres can be contactedwith a silanizing agent to form crush resistant microspheres having acompletely silanized surface. The silanized microspheres exhibitenhanced chemical stability with respect to hydrolysis.

Microspheres of the present invention demonstrate high mechanicalstability when used in columns for high pressure liquid chromatography.It is believed that the stability results from a portion of the silicabeing dissolved by water containing HF or a basic activator. Theresulting silica is reprecipitated at points of contact between thecolloidal particles making up the aggregate structure of the poroussilica microspheres. Thus, the reprecipitated silica provides additionalreinforcement to the structure of the silica microspheres.

I. Heat Strengthened Thermally-Dehydroxylated Porous Silica Microspheres

Heat strengthened thermally-dehydroxylated porous silica microspherescan be prepared according to a method similar to that described in U.S.Patent 3,782,075, the disclosure of which is incorporated herein byreference. An aqueous sol of silica is formed and mixed with acopolymerizable mixture of urea and formaldehyde or melamine andformaldehyde. Polymerization is initiated and coacervation of theorganic material into microspheres containing the colloidal particlesoccurs. The microspheres are then solidified, collected, washed anddried. At this stage, the microspheres consist of a plurality ofcolloidal particles embedded in a sphere filled with polymer. Theorganic material is then burned off at a temperature sufficient tooxidize the organic constituents without melting the inorganic material.Generally, the organic material is burned off at about 550° C. Theporous microspheres are then sintered at an elevated temperature for atime sufficient to strengthen the microparticles to the point where theywill not fracture in use. A good indication of whether enough sinteringhas occurred is when the specific surface area of the microspheres hasbeen reduced to a value which is at least 10% less than the surface areaof the colloidal particles themselves.

Formation of the microspheres proceeds by association of the inorganiccolloidal particles with the organic coacervate. It is postulated thatthe extreme uniformity in both the size of the microspheres and thedistribution of the colloidal particles within the microspheres dependon an interaction between hydroxyl groups on the surface of thecolloidal particles and portions of the organic polymer chains. For thisreason, at least prior to the onset of polymerization, the colloidalparticles must have hydroxyl groups on their surface equivalent to ahydrated oxide surface.

The ultimate particles of the present invention must be colloidal insize. This means that at least two of the dimensions of these particleswill be in the range of 3 to 500 nm and the other dimension will be inthe range of 3 to 1000 nm. Particles having one dimension greater than aμm or having any dimension greater than about 0.1 times the diameter ofthe microspheres are difficult to incorporate into sphericalmicroparticles since the large dimension interferes with the formationof discrete spherical units.

The organic components used to form the microspheres must be initiallysoluble in water and miscible with the silica colloid withoutflocculating or dissolving it at the pH at which the reaction occurs.The polymer when formed must be insoluble in water. While a variety oforganic materials are suitable, it appears that the highest degree ofuniformity in both particles size and pore size distribution occurs whena copolymerizing mixture of urea and formaldehyde or melamine andformaldehyde is used. Urea and formaldehyde in molar ratio of about 1 to1.2 or 1.5 and a pH or about 1.0 to 4.5, and melamine and formaldehydein molar ratio of about 1 to 3 and a pH or about 4 to 6 are suitable.

The ratio of organic material to silica should be such that afterpolymerization, the precipitated particles contain about 10 to 90 weightpercent of silica. Expressed in terms of volume, the percent volume ofinorganic material should range from about 10 to about 50. To obtaincoherent porous spheres after the organic matter is burned out, theremust be a sufficiently high concentration of silica particles within thematrix to link together into a three-dimensional matrix. This networkmay be very fragile, when obtained at 550° C., but if heated undisturbedat higher temperature to initiate sintering, the porous microspheresdevelop strength. To insure that sufficient sintering has occurred toprovide the desired strength, the particles are generally sintered at atemperature, usually above 900° C., which is sufficiently high to reducethe specific surface area of the particle by at least 10% below thevalue for the colloidal particles from which they are formed. Themicrospheres have uniform pores, the diameters of which depend on thesize of the colloidal particles used in their preparation and the volumeratio of the organic polymer to the silica material used. The larger thecolloidal particles, the larger the pores between them, and the greaterthe proportional volume of organic polymer in the microspheres whenformed, the more open the network of silica particles and the wider thepores.

Calcining porous silica microspheres has two effects. First, theultimate particles making up the porous structure sinter or fusetogether to some extent at their points of contact to increase thephysical strength of the microspheres. Second, the hydroxylated surfaceof silanol groups present before being heated is dehydroxylated, i.e.,water is lost by condensation of neighboring SiOH groups, generallyleaving most of the surface consisting of siloxane groups, SiOSi.Generally, these siloxane groups are inert to reaction with silanizingagent. It has been found that the resulting microspheres have a totalconcentration of silanol groups of substantially less than about 5.5μmol/m². It has been found that the microspheres can be rehydroxylatedto provide an improved chromatographic material and a good precursor forsubsequent reaction with silanizing agents.

Methods that have been used to rehydroxylate silica surfaces includeboiling the calcined silica particles in water for extended periods oftime or in dilute nitric acid for several hours. Under these conditions,some rehydroxylation occurs and typically there are then about threesilanol (SiOH) groups on the surface per square nanometer, or about 5μmol/m². This has been the practical limit of these processes and ischaracteristic of commercially available calcined/rehydroxylated silicacolumn packings. Another known method of rehydroxylation involvedhydrothermal treatment with steam. This aggressive techniquesignificantly degrades the porous structure of the silica particles andleaves a surface that is poorly suited for chromatography.

II. Porous Silica Microspheres Having an Enriched Surface Concentrationof Silanol Groups

The porous silica microspheres having a total concentration of silanolgroups of from about 6 to about 16 μmol/m² can be prepared by contactingheat strengthened thermally-dehydroxylated porous silica microsphereswith water in the presence of HF or at least one basic activatorselected from the group consisting of quaternary ammonium hydroxides,ammonium hydroxide, and organic amines. The contacting normally isconducted at a temperature of from about 25° C. to about 100° C. forsufficient time to generate the desired surface concentration of silanolgroups. The strength characteristics of the resulting microspheres aresuperior to those of the heat-strengthened microspheres described inSection I above. The strength is derived from the inherent integrity ofthe silica particles based on the optimum ratio of pore volume to silicavolume, the calcination pretreatment, and the addition of silica at thecontact point of the colloidal particles comprising the microspheresduring the rehydroxylation process.

The concentration of silanol groups on a silica surface can bedetermined in several ways including infrared spectroscopy, solid-statemagic angle spinning nuclear magnetic resonance, proton-spin countingNMR, and/or thermogravimetric analysis, the latter generally beingpreferred because of its simplicity and precision. It is noted in thisconnection that excessive rehydroxylation of a silica surface to greaterthan about 8 μmol/m² of silanol groups will result in silanol groupsthat are "buried" beneath the silica surface. These groups are detectedby TGA, but generally are not available for chromatographic interactionsor for reactions with silanizing agents to form bonded-phase packings.

It has been found that activators which promote rehydroxylation to thedesired total concentration of silanol groups of from about 6 to about16 μmol/m² are HF and basic activators selected from the groupconsisting of quaternary ammonium hydroxides, ammonium hydroxide, andorganic amines. Preferably, the basic activator is selected from thegroup consisting of tetraalkylammonium hydroxide, ammonium hydroxides,primary organic amines and secondary organic amines. The relative rateof dissolution of silica by a basic activator can be controlled bymaintaining pH in the weakly-basic range. Most primary and secondaryorganic bases rapidly dissolve silica above a pH of about 10.5. The rateis much slower below this pH value. A basic activator that provides abuffered pH of about 10.5 in dilute solution has desirable properties,especially when hydroxylation is carried out at 25°-50° C. At thesetemperatures the solubility and the rate of transfer of silica is muchlower than at higher temperatures such as 100° C. Preferably, a basicactivator is added in a sufficient amount to generate a pH of from about9 to about 10.5.

For basic activators the overall rate of attack on the silica surfacegenerally decreases from methyl to ethyl to propyl. For example, normalethyl-, propyl-, and butylamine, secondary ethyl-propyl- and butylamineare effective activators. Monomethyl- and dimethylamine can be utilized,if care is exercised. Steric effects appear to have a noticeableinfluence on the dissolution rate of the silica gel latice as disclosedby A. Wehrli, J. C. Hildenbrand, H. P. Keller, R. Stampfli, R. W. Frei,J. Chromatogr., 149:199 (1978). Methyl amines can be less practicalbecause of their strong tendency to attack silica. Thus, methyl aminesare more difficult to control in generating the desired concentration ofsilanol groups. It has been found that the rate of attack of a base onsilica is dependent on the strength (pK_(B) value), concentration, andgeometry of a selected basic activator.

Although tetraalkylammonium hydroxides show strong aggressiveness fordissolving silica, these compounds are preferred basic activators forrehydroxylation. This is the case even though tetramethylammonium,tetrapropylammonium and tetrabutylammonium hydroxide show equal or aneven greater tendency than alkali hydroxides to attack the silicasurface. Tetraalkylammonium hydroxides are effective activators becauseat a pH of from about 9 to about 10.5, very little of the free baseremains in solution. It is believed that most of the base is absorbed asa monolayer on the silica surface, making the silica somewhathydrophobic. Hydroxyl ions remaining in solution catalyze the breakingof siloxane groups, while the monolayer of activator on the silicasurface retards dissolution and deposition of silica. Therefore, theprocess can be conveniently interrupted before the degree ofhydroxylation passes beyond the desired range.

Tetrabutylammonium hydroxides, ammonium hydroxide and primary organicamines are preferred basic activators. When a sufficient amount of thesebases is added to an aqueous suspension of microspheres to raise the pHto a value between 9 and 10.5, very little free base remains insolution. Most of the base is adsorbed as a monolayer on the silicasurface making the silica surface somewhat hydrophobic. Hydroxyl ionsremaining in solution catalyze the breaking of siloxane groups while themonolayer of activator on the silica surface retards dissolution anddeposition of silica. This process can be stopped before rehydroxylationof the microspheres passes beyond the desired concentration of silanolgroups. Most preferably, the primary and secondary amines containhydrocarbon groups that retard dissolution of silica.

Ammonium hydroxide is also a preferred basic activator. Dilute ammoniumhydroxide at pH 10 reacted with silica for 18 hours and 25° C. is apreferred method for rehydroxylating a silica surface to the desiredconcentration of silanol groups. Hydrolysis of a 440 m² /g silica bythis procedure changed the surface area by only about 25%, and the porevolume of the silica remained essentially unchanged.

Most preferably, the basic activator is at least one primary amineselected from the group consisting of ethylenediamine, n-propylamine andn-butylamine. These amines can generate a pH of from about 9 to about10.5. A pH in this range accelerates rehydroxylation of the silicasurface, without significant change in the surface area and porediameter of the silica structure as can occur with strong organic basessuch as quaternary ammonium hydroxides. When the latter are used asactivators, their concentration should be low and the initial pH shouldnot exceed about 10. Secondary amines such as diethyl-, dipropyl-, anddibutylamine are also suitable activators but rehydroxylation reactionsare generally slower. Tertiary amines are less preferred activators.

Alkali- or alkaline-earth hydroxides such as NaOH, KOH and CaOH aredifficult to control in the rehydroxylation process. Use of these agentscan result in significant undesirable changes in the pore structure andsurface area of the starting silica. In addition, use of these agentsresults in an undesired contamination of the starting silica with thecation from the hydroxide. This contamination causes deleterious effectswith the silica support in subsequent chromatographic uses.

Acidic solutions of ionic fluorides are also suitable activators.Suitable sources of HF are HF, NH₄ F and other ionic fluorides notcontaining a metal or metalloid cation which could deleteriouslycontaminate the highly purified silica. These activators can be added toan aqueous solution containing thermally dehydroxylated microspheresaccording to the following procedures. The aqueous solution is adjustedto a pH of about two to about four with a mineral acid such ashydrofluoric, hydrochloric or sulfuric acid. A suitable source of freeHF is added to the solution in a concentration that acts as a catalyticagent for the dissolution of the silica surface. The preferredconcentration of HF is a function of the surface area of the silica.Preferably, microspheres of the present invention are rehydroxylated inthe presence of free HF in a concentration of from about 50 to about 400ppm. Typically, HF in a concentration of from about 200 to about 400 ppmis suitable to activate the rehydroxylation of a 300-400 m² /g silica.It is believed that fluoride, introduced as HF or an ionic salt thereofat a pH from about 2 to about 4, reacts with a small amount of dissolvedsilica to form SiF₆ ⁻². The SiF₆ ⁻² remains in equilibrium with a lowconcentration of HF. This system functions as an activator to increasethe rate of silica hydroxylation.

III. Silanized Porous Silica Microspheres

Porous silica microspheres having a completely silanized surface can beprepared from the microspheres having a total concentration of silanolgroups of from about 6 to about 16 μmol/m². The microspheres having anenriched surface concentration of silanol groups prepared in Section II,above, are contacted with a silanizing agent at a temperature of fromabout 25° to about 100° C. for sufficient time to generate a completelysilanized surface. Suitable silanizing agents are disclosed above andknown in the art. A partial list of suitable silyl groups includestrimethyl-, dimethylbenzyl-, dimethylbutyl-, dimethyloctyl-,dimethyloctadecyl, dimethyl-3-cyanopropyl-,dimethyl-3-glycidoxylpropyl-, and methyldiphenylsilyl. Other suitablesilanizing agents are disclosed in U.S. Pat. Nos. 3,722,181 and3,795,313, the disclosure of which are incorporated herein by reference.

UTILITY

In optimum dimensions, microspheres of the present invention exhibitssuperior performance in various forms of liquid chromatographicapplications including bonded-phase, liquid-solid and size-exclusion.For example, highly efficient liquid-solid chromatography can be carriedout with microspheres having a diameter in the 1.0 to 15.0 μm range madefrom colloidal particles in the 5 to 50 nm range. High speedbonded-phase packings can be prepared by coating microspheres having adiameter in the 1.0 to 15.0 μm range and made from colloidal particlesin the 50 to 100 nm range, with appropriate covalently bonded organicligands or with polymerized coatings. These particles can also bereacted with ion-exchange media to produce supports for ion-exchangechromatography. Highly efficient gas-liquid and gas-solidchromatographic separation also can be carried out with microsphereshaving a diameter in the range of 20 to 30 μm, made from colloidalmicroparticles in the 5 to 200 nm range. The range of useful microspherediameters extends from about 0.5 to 35 μm.

Since the microspheres prepared from each size of colloidal particlesconsists of a totally porous structure having a narrow range of poresizes, by varying the size of the colloidal particles, microsphereshaving a predetermined range of relatively homogeneous pore sizes can beproduced. Silica microspheres with pores of known dimension can be usedfor high speed size-exclusion chromatographic separation such as gelpermeation and gel filtration. These separation techniques are based onthe differential migration of molecules based on molecular size ormolecular weight considerations. Small particle size promotes rapid masstransfer so that mobile phase velocities much higher than normal can beused while still maintaining equilibrium in the diffusion-controlledinteraction that takes place within the pores in the totally porousstructure. The strong, rigid characteristics of the present microspherespermit their use at very high pressures without particle degradation ordeformation. The spherical nature of the particles permits the packingof columns with a large number of theoretical plates, which is ofparticular importance in the separation of large molecules. Of primeconsideration in the size-exclusion chromatographic process is theinternal volume of the particles used in the separation. Pore volume ofthe particles is moderately high in the microspheres, usually from 50 to65% (measured by N₂ adsorption with the B.E.T. method) which iscomparable to that found for the porous glasses and the porous organicgels widely used for size-exclusion chromatography.

The silica microspheres are also useful in gel filtration separations inaqueous systems and for the separation of small polar molecules.Microspheres having pores in the 50 to 2500 Angstroms range permit thehigh-speed size-exclusion chromatographic separation of a large varietyof compounds in both aqueous and nonaqueous systems.

One of the factors that affects efficiency is the nature of packingformed in a column or structure which constitutes the resolving zone.One advantage of the microspheres of the present invention is that theirhigh mechanical strength and spherical and uniform size permits ease ofpacking into a dense bed. A common column packing method is dry packing.However, when the particles are less than about 20 μm in diameter,high-pressure wet-slurry packing must be used. The uniform porous silicamicrospheres of this invention can be easily and convenientlyhigh-pressure slurry-packed into columns after producing a stablesuspension. The suspension of particles is accomplished by techniquesdescribed in L. R. Snyder and J. J. Kirkland, "Introduction to ModernLiquid Chromatography", Second Edition, John Wiley and Sons, Inc. 1979,p. 207. Chromatographic columns herein described were prepared from suchslurries according to a procedure similar to that described by L. R.Snyder and J. J. Kirkland, at p. 210.

The porous silica microspheres of the present invention demonstratehigher permeability (less resistance to flow) than irregularly-shapedand wider size range silica particles of the same size. Pressurerequirements for microsphere columns are sufficiently low so as to behandled by most of the pumps currently being used in liquidchromatography. One-meter long microsphere columns of 5 to 6 μmparticles can be operated at carrier velocities of 0.5 cm/sec withpressures of only about 2400 psi (16.56 kPa). Such a column wouldexhibit >60,000 theoretical plates, which should permit very difficultseparations.

The present invention is further described by the following exampleswherein all parts and percentages are by weight and degrees are Celsius.In the Examples, pH measurements of silica suspensions were carried outwith a Beckman 43 pH-METER equipped with automatic temperaturecompensation and a Beckman refillable combination electrode. Theelectrode was calibrated with pH 4, pH 7 and pH 10 standard solutions,depending on the pH range investigated. The silica suspensions wereprepared by adding 50 g of deionized water to 1 g of silica. Afterstirring for 2 minutes, the pH value of the suspension and the time ofmeasurement were recorded. The pH values were determined after at least10 minutes of equilibration. Thermogravimetric analysis andchromatographic results shown in the Examples were conducted accordingto the following procedures.

Thermogravimetric Analysis

TGA-measurements were conducted with a Model 990 TGA-analyzer (E. I. duPont de Nemours & Company, Wilmington, DE) according to the followingprocedure. 20 to 100 mg of silica were loaded into a small quartzcrucible and placed in the TGA-analyzer. The resulting samples wereheated to 120° at a rate of 10°/min while dry nitrogen gas was passedthrough the heating chamber at a flow rate of 50 mL/min to removephysically adsorbed water from the silica surface. The samples weremaintained at 120° until no further weight loss could be observed. Thetemperature was then increased to 300° at the same heating rate asbefore, and held at this temperature until a constant weight wasreached. The same procedure was repeated at 500°, 700°, 900°, 1050°, and1200°. At each temperature, a characteristic weight loss could beobserved for each sample.

The total concentration of silanol groups on the silica was calculatedfrom the total percent weight loss found at 1200° following the dryingstep at 120° C. The calculation of SiOH concentation was based on theassumption that two moles of SiOH groups combine on heating to form onemole of water which is lost from the sample during the heatingprocedure. The total concentration of silanol groups on the silica wascalculated according to the following formula: ##EQU1## where W is thepercent weight loss difference at equilibrium from heating at 120° tothe heating at 1200°, and SA is the BET nitrogen surface area of thesilica in m² /g.

A relatively pure silica sample begins to soften above 1000°.Significant weight loss can be detected after 1 hour of heating attemperatures greater than 1000° with some samples. To ensure that thisobservation was not due to an artifact (e.g., formation of silicanitride), experiments were repeated with argon as the purging gas. Nodifferences in the TGA-curves could be detected. Thus, the weight lossupon heating is due to the loss of chemically bonded water from thesilica structure.

Control experiments (no sample) showed no apparent weight loss attemperatures above 1000°, indicating no significant response due tobuoyancy effects at these high temperatures. Also, the observed weightloss at about 1000° is not due to desorption of gas during the sinteringof the silica, since BET (Brunauer, Emmett and Teller) measurementsrevealed that only extremely small amounts of gases are adsorbed on thesilica at high temperatures (e.g., 380°).

Chromatographic Procedures

Stainless steel column blanks, 150 mm long and 4.6 mm inner diameterwith mirror-finished walls were used. Low dead-volume stainless steelcompression fittings with metal screens retained the packing. For asingle column, 2 to 3 g of silica was suspended in 14 mL ofhexafluoroisopropanol slurrying liquid. Hexane was used as pressurizingliquid at 10,000 psig (69.0 kPa). Columns were packed according to amethod similar to that described in, L.R. Snyder and J. J. Kirkland,"Introduction to Modern Liquid Chromatography", 2nd edition, John Wiley& Sons, New York, 1979, Chapter 5. Prior to chromatographic testing,columns were carefully purged with isopropanol and methanol.

Chromatographic experiments were performed with a Du Pont 8800 LCinstrument equipped with column oven, Rheodyne injection valve and a DuPont 860 Absorbance Detector or Du Pont 862 UV SpectrophotometerDetector. Solvent containers were stored in well-ventilated areas, andall mobile phases were carefully degassed by helium purge before use.All columns were thermostated at 50°. In the Examples, the followingtest-mixtures were used:

(1) Test Mixture A contained 10 μL of 1-phenylheptane+10 μL of1-phenylhexane in 4 ml of methanol.

(2) Test Mixture B contained 25 μL of a polarity mixture in 4 ml ofmethanol. The polarity mixture contained 250 μL of 5-phenylpentanol, 10μL of N,N-diethylaniline, 50 μL of 2,6-di-t-butylpyridine and 1000 μL of1-phenylheptane.

Injections of 5 to 10 μL were used to produce chromatographic peaks on a1 mV recorder at 254 nm detection wavelength and an attenuation of 0.05.

New columns were first tested with Test Mixtures A and B usingmethanol/water eluents (80/20, 70/30 or 60/40). Retention times,k'-values and column plate counts for the different peaks weredetermined for each chromatogram. The relative retention or selectivityfactor of the basic probe, N,N-diethylaniline, to the neutral compound,1-phenylheptane (capacity factor k₁ '/k₂ ' ratio) was used to indicatethe adsorptivity of column packings.

EXAMPLE 1 Preparation of Porous Silica Microspheres Having aSilanol-Enriched Surface

13 g of heat strengthened thermally-dehydroxylated porous silicamicrospheres having a surface area of 443 m² /g, an average porediameter of 77 Angstroms and a total silanol concentration of no morethan 5.9 μmol/m², which are available commercially from E. I. du Pont deNemours and Company under the registered trademark Zorbax-PSM-60 (5μm),were heated at 850° for 3 days. The resulting silica was placed into a250 mL 3-neck pyrex flask equipped with a reflux condenser andheater-stirrer and suspended in 130 mL of water containing 200 ppm of HF(400×10⁻⁶ liter of a 50% HF-solution in 1 L of deionized water). ThepH-value of the resulting suspension was 3. The suspension was boiledfor 3 days, allowed to cool in the reaction flask to ambienttemperature, and then filtered using an extra-fine fritted disk. Theresulting filtrate exhibited a pH-value of 3. By washing the silica with2000 mL of deionized water the pH-value of the filtrate was increased to6. The silica was rinsed with acetone and dried at 120° and 0.1 mbar(0.01 kPa) for 15 hours. The silica was then rinsed successively with300 mL of a water/ammonium hydroxide-solution (pH=9), water toneutrality, and 100 mL of acetone and dried at 0.1 mbar and 120° for 15hours. 1 g of the resulting silica suspended in 50 g of water exhibiteda pH-value of 5.3 as compared to a pH value of 4.1 for the startingsilica. The rehydroxylated silica had a surface area of 347 m² /g, anaverage pore diameter of 80 Angstroms and a total silanol concentrationof 9.0 μmol/m² by TGA.

EXAMPLE 2 Preparation of Porous Silica Microspheres Having aSilanol-Enriched Surface

13.5 g of the silica starting material described in Example 1 wereheated at 850° for 3 days. The resulting silica was placed into anapparatus similar to that described in Example 1 and suspended in 200 mLof deionized water. The resulting suspension was adjusted to a pH valueof 9 with tetrabutylammonium hydroxide solution. The resultingsuspension was then heated to 100° for 26 hours, allowed to cool toambient temperature, and filtered using an extra-fine fritted disk. Theresulting silica was washed to neutrality with 1000 mL of water. Theresulting silica powder was then rinsed with 300 mL of acetone and driedfor about 18 hours in a vacuum oven at 120° and 0.1 mbar (0.01 kPa). 1 gof resulting silica suspended in 50 g of water showed a pH-value of 5.6after 10 minutes. To ensure that no tetrabutylammonium ion was adsorbedto the surface, the silica was washed with 200 mL of diluted nitric acid(1 mL of concentrated HNO₃ in 200 mL of water) and another 1000 mL ofdeionized water to neutrality. After washing with 300 mL of acetone andrepeating the drying procedure, the pH-value of the silica was measuredagain. No change of the original pH-value of 5.6 was observed. Theresulting silica had a surface area of 356 m² /g, an average porediameter of 87 Angstroms, and a total silanol surface concentration of9.1 μmol/m² by TGA.

EXAMPLE 3 Preparation of Porous Silica Microspheres Having aSilanol-Enriched Surface

15 g of the silica starting material described in Example 1 were heatedat 850° for 3 days. The resulting silica was placed into an apparatussimilar to that described in Example 1 and suspended in 150 mL ofdeionized water. The resulting suspension was adjusted to pH 9 by theaddition of ethylenediamine. The suspension was heated at reflux for 24hours, allowed to cool to ambient temperature, and filtered using anextra-fine fritted disk. Refluxing was performed under an argonatmosphere to avoid a reaction of ethylenediamine with carbon dioxide.The resulting sample was washed in nitric acid, deionized water, andacetone according to a method similar to that described in Example 2. 1g of the resulting silica suspended in 50 g of water exhibited apH-value of 5.3. The silica had a surface area of 224 m² /g, an averagepore diameter of 142 Angstroms, and a total silanol concentration of 9.9μmol/m² by TGA.

EXAMPLE 4 Preparation of Porous Silica Microspheres Having aSilanol-Enriched Surface

15 g of the silica starting material described in Example 1 weresuspended in distilled water and the resulting mixture was adjusted to apH of 10 with ammonium hydroxide. The mixture was allowed to stand for18 hours at room temperature and filtered. The resulting sample waswashed with 500 mL of distilled water, 200 mL of nitric acid (1 mL ofconcentrated nitric acid in 200 mL of water), and 500 mL of distilledwater to neutrality. The sample was washed with 200 mL of acetone,air-dried, and then dried in a vacuum oven at 100° for 16 hours. Theresulting hydroxylated silica exhibited a pH value of 4.8, a nitrogensurface area of 381 and 387 m² /g (duplicate analysis) and an averagepore diameter of 76 and 80 Angstroms (duplicate analysis) as compared tothe starting silica which exhibited a pH value of 4.1, surface area of443 m² /g and an average pore diameter of 77 Angstroms.Thermogravimetric analysis of the hydroxylated silica showed a totalsilanol concentration of 8.9 μmol/m² by TGA.

EXAMPLE 5 Preparation of Wide-Pore Silica Microspheres Having aSilanol-Enriched Surface

15 g of heat strenghthened thermally-dehydroxylated microspheres, whichare available commercially from E. I. du Pont de Nemours and Companyunder the registered trademark Zorbax-PSM-300, were placed in a quartzdish and heated in a nitrogen-purged furnace at 200° for 8 hours, at400° for 15 hours, and at 850° for 3 days. The resulting initial sampleexhibited a nitrogen surface of 56 m² /g and an average pore diameter of442 Angstroms by nitrogen adsorption and 338 Angstroms by mercuryintrusion. The sample was placed into a 250 mL three-neck glass flaskequipped with a reflux condenser and a heater-stirrer, and suspended in150 mL of water containing 75 ppm of HF. The resulting mixture wasboiled for 3 days, allowed to cool in the reaction flask to ambienttemperature, and filtered using an extra-fine fritted disk. Theresulting solid was washed with water to neutrality (about 600 mL) andheated at 100° in distilled water for 10 hours. The resulting mixturewas filtered and the resulting solid was washed with 200 mL of acetoneand dried at 120° and 0.1 mbar (0.01 kPa) for 15 hours. The resultingsilica had a nitrogen surface area of 57 m² /g, an average pore diameterof 289 Angstroms by nitrogen adsorption and a total silanolconcentration of 15.6 μmol/m² by TGA, as compared to the starting silicawhich exhibited a total silanol concentration of 5.8 μmol/m².

EXAMPLE 6 Preparation of Porous Silica Microspheres Having a SilanizedSurface

This Example was carried out in an Edwards high vacuum system and asilylation apparatus similar to that described in A. Haas et al.,Chromatographia, 14:341 (1981) and G. Schomburg et al., Chromatog. J.,282:27 (1983), the disclosures of which are incorporated herein byreference. 15 g of the porous silica microspheres having asilanol-enriched surface prepared in Example 1 were dried in thereaction chamber of the silylation apparatus at 200° and 2×10⁻⁶ mbar for24 hours and allowed to cool to ambient temperature. 30 mL oftrimethylsilylenolate were placed in a dropping funnel, under an argonatmosphere. The funnel was evacuated, and the enolate allowed to comeinto direct contact with the silica. As the reaction proceeded, bubblesof acetylacetone were released. After 1 hour, the silica was heated to60° for another 4 hours. The resulting product was washed with 200 mLportions of dry toluene, dichloromethane, methanol, methanol-water(1:1), and acetone, successively. This procedure produced atrimethylsilyl concentration of 4.0 μmol/m² as determined by elementalanalysis. The silica was tested as a chromatographic material using TestMixture A and Test Mixture B, previously described herein. The testswere conducted using a methanol/water eluent (70/30), a flow rate of 1mL/min and a pressure of 725 psi (5000 kPa). The results of theseseparations are shown in FIG. 1.

The stability of the trimethylsilyl-modified rehydroxylated microspheresprepared in this Example was compared to that of the startingmicrospheres described in Example 1. In these tests degradation of thetrimethylsilyl-modified silica was initiated by purging columns of thechromatographic materials with water. Periodically during this purging,these columns were tested chromatographically with the test-probesamples described under "Chromatographic Procedures". The tests wereconducted using a methanol/water eluent (60/40), a flow rate of 1 mL/minand a T₀ of 1.68 min. The results for the microspheres shown in Table 1using the specified test components are shown in FIG. 2.

                  TABLE 1                                                         ______________________________________                                                Silica                                                                Test    Description      Test Compound                                        ______________________________________                                        A       Dehydroxylated micro-                                                                          1-Phenylhexane                                               spheres described in                                                          Example l                                                             B       Dehydroxylated micro-                                                                          N,N--Diethylaniline                                          spheres described in                                                          Example 1                                                             C       Trimethylsilyl-  1-Phenylhexane                                               modified microspheres                                                         prepared in Example 7                                                 D       Trimethylsilyl-  N,N--Diethylaniline                                          modified microspheres                                                         prepared in Example 7                                                 ______________________________________                                    

The results show that with water purge, the retention of the neutraltest compound, 1-phenylhexane, as measured by the capacity factor, k',decreased with increasing column volumes of water purged through thecolumn. The decrease in k' for the trimethylsilyl-modifiedrehydroxylated microspheres prepared in this Example degraded by thisprocedure was significantly less than that of the starting microspheres.More significantly, the column prepared from the starting microspheresshowed an increased retention for the basic probe, N,N-diethylaniline,while the column packing prepared from the microspheres of this Exampleinitially showed decreased retention (indicating less-binding toresidual acidic sites initially on the packing), and only a slightincrease in k' values with water purge, even with the passage of morethan 11,000 column volumes (more than 18,000 mL) of water.

These results show the improved stability of the bonded-phase packingmade from the hydroxylated silica of this invention, and thesignificantly reduced adsorption of basic probes to the packingmaterial, even when a substantial concentration (more than 3/4) of thetritmethylsilyl groups had been hydroxylated from the surface, asmeasured by the decrease of k' values from the initial point (no waterpurge).

EXAMPLE 7 Preparation of Porous Silica Microspheres Having a SilanizedSurface

10 g of the silanol-enriched microspheres prepared in Example 3 weresilanized substantially according to the enolate reaction described inExample 6. The final product exhibited a trimethylsilyl coverage of 3.78μmol/m² as determined by elemental analysis. Capacity factor (k') valuesfor 1-phenylheptane and N,N-diethylaniline were k'₁ =2.50 and k'₂ =0.82,respectively, using a chromatographic mobile phase of 60/40methanol/water. The selectivity factor, k'₁ /k'₂, was 3.05. Thesechromatographic data indicate low adsorption for the basic solute,N,N-diethylaniline, and normal retention for 1-phenylheptane. Tests withwater purging according to a procedure similar to that employed inExample 6 indicated increased stability of the trimethylsilyl group ofthis hydroxylated silica, as compared to the starting microspheresdescribed in Example 1.

EXAMPLE 8 Preparation of Porous Silica Microspheres Having a SilanizedSurface

10 g of the silanol-enriched microspheres prepared in Example 2 weresilanized by the enolate reaction in the manner described in Example 6.The final product exhibited a trimethylsilyl concentration of 4.42 μmolm² /g, as measured by elemental analysis. Using the chromatographic testprocedure employing methanol/water in a ratio of 70/30, the capacityfactor, k', values of k'₁,=7.35 and k'₂ =1.80 were obtained for1-phenylheptane and N,N-diethylaniline, respectively, with a selectivityfactor, k'₁ /k'₂, of 4.08 in this test. These results indicated reducedabsorption of the basic test compound, N,N-diethylaniline, relative tothe thermally-dehydroxylated microspheres described in Example 1.Improved stability of this bonded-phase material was also indicated inthe water-purge test.

EXAMPLE 9

Preparation of Porous Silica Microspheres Having a Silanized Surface

10 grams of the silanol-enriched microspheres prepared in Example 4 weresilanized substantially according to the enolate reaction described inExample 6. The trimethylsilyl group concentration on the resultingsilica was 3.89 μmol/m², as measured by elemental analysis.Chromatographic tests indicated a low order of adsorption for the basicprobe, N,N-diethylaniline. Tests with water purging according to theprocedure employed in Example 6 indicated improved stability of thetrimethylsilyl bonded-phase material, relative to the startingmicrospheres described in Example 1. FIG. 3 shows the chromatographicseparation of Test Mixture B using a methanol/water eluent (70/30), aflow rate of 1 mL/min, and a pressure of 725 psi (5000 kPa).

EXAMPLE 10 Preparation of Wide-Pore Silica Microspheres Having aSilanized Surface

15 grams of the rehydroxylated microspheres prepared in Example 5 weredried for 30 hours at 200° and 0.1 mbar under an argon atmosphere. Thepowder was then suspended in a 100 mL of toluene (HPLC-grade) in a200-mL 3-neck flask fitted with a reflux condenser and an argon purgingsystem. To this mixture was added 250 mL of trimethylchlorosilane and4.09 g (or 50 μmol) of pyridine (99.9%). This mixture was heated at 120°in an oil bath for 65 hours. The silica was then transferred onto a fineporous frit and washed with 200 mL of toluene, 200 mL of cyclohexane,200 mL of dichloromethane, 200 mL of methanol, 200 mL of methanol/water,3:1, and finally, 200 ml acetone. After filtration the powder was driedin a vacuum oven at 120° and 0.1 mbar for 30 hours. This final producthad a trimethylsilyl concentration of 3.96 μmol/m², based on elementalanalysis.

FIG. 4 shows a comparison of chromatographic separations for thisproduct (Silica A) versus a column of the trimethylsilyl-modifiedmicrospheres initial starting silica used in Example 5 (Silica B). Thetests were conducted using a methanol/water eluent (60/40) at a flowrate of 1 mL/min and pressures of 725 psi (5000 kPa) and 1160 psi (8000kPa), respectively. The chromatogram for the packing made from thehydroxylated product of this Example shows earlier elution of the basicprobe, N,N-diethylaniline, with excellent peak shape, indicating noundesired adsorption of this material. On the other hand, the column ofthe silanized silica exhibited strong adsorption of N,N-diethylaniline;in addition, N,N-diethylaniline eluted as a very broad tailing peak,indicative of unwanted adsorption. The data in FIG. 4 also indicatesstrength of the microsphere was improved, as indicated by the lowercolumn back pressure exhibited for this slurry-packed material, relativeto the starting silica described in Example 5.

The efficacy of the chromatographic columns prepared in this Example isfurther demonstrated in FIG. 5, which shows separation of a mixturecontaining the basic peptide mellitin (molecular weight=2600; 26 aminoacids) on a column of the packing prepared in this example. Theseparation was conducted with a 60 min gradient starting with 20%acetonitrile in water containing 0.1% trifluoroacetic acid and endingwith 100% acetonitrile containing 0.1% trifluoroacetic acid (v/v%). Theflow rate was 1.0 mL/min and the temperature was 35°. The results showthat all of the compounds are successfully eluted and separated bygradient elution on the silanized microspheres of this Example. Elutionof the highly basic peptide, mellitin, could not be achieved when usingalkyl bonded-phase columns prepared from silicas that were not fullyhydroxylated.

EXAMPLE 11 Crush Resistance of Porous Silicas

A comparision of crush resistance of the porous silicas shown in Table 2was conducted on an Instron Model 1127 Universal Testing Machine.

                  TABLE 2                                                         ______________________________________                                        Test     Silica Description                                                   ______________________________________                                        A        Silanol-enriched microspheres prepared in                                     Example 1.                                                           B        Heat strengthened thermally-dehydroxylated                                    microspheres described in Example 1.                                 C        Porous silica commercially available from                                     Macherey and Nagel Company, Duren, FRG                                        under trademark Nucleosil ™.                                      D        Porous silica commercially available from                                     Separations Group, Hesperia, California                                       under the trademark Vydac ™.                                      ______________________________________                                    

Crush resistances for the specified silicas were determined according tothe following procedure. One gram of each porous silica was loaded intoa stainless steel die normally used for preparing potassium bromidedisks for infrared spectroscopy studies. The die had a one-half-inchdiameter piston used to form the disks by high-pressure loading. Theresulting silica samples were introduced into the die and loaded at apiston travel rate of 0.01 in/min. All samples were initially compacted(or pre-loaded) to a firm homogenous bed with a loading of 250 kg. Thesamples were then continuously loaded to a total pressure of 14,500 psi(1.00×10⁵ kPa). The results are shown in FIG. 6. In the FIG. a steepcurve represents the ability of stronger particles to readily accept thepressure load; the pressure increases rapidly as crush-resistantparticles are loaded. Conversely, a less steep curve indicates that theparticles are crushing more readily since the pressure increases moreslowly as particles crumble under the load. Data in this curve show thatone of the silanol-enriched microspheres prepared in Example 1 showedthe highest crush resistance of any silica tested.

The improved crush resistance of the hydroxylated microspheres isbelieved to be based on the fact that, during the hydroxylationreaction, silica is dissolved and reprecipitated at the points ofcontact of the colloidal particles making up the aggregate structure.Thus, this fully hydroxylated, reprecipitated silica further binds thecolloidal particles together within the aggregate structure, increasingthe strength of the microsphere.

What is claimed is:
 1. A chromatographic material comprising improvedporous silica microspheres having an average diameter of about 0.5 toabout 35 μm, substantially all of said microspheres having a diameterranging from about 0.5 to about 1.5 times said average diameter; saidmicrospheres consisting essentially of a plurality of substantiallyuniform-size colloidal particles, having a silica surface, arranged inan interconnected three-dimensional lattice; said colloidal particlesoccupying less than about 50 volume percent of said microspheres withthe remaining volume being occupied by interconnected pores havingsubstantially uniform pore size distribution; said microspheres having atotal concentration of silanol groups from about 6 to about 16 μmol/m².2. A chromatographic material as defined in claim 1 wherein themicrospheres have an average diameter of from about 1.0 to about 10 μm.3. A chromatographic material comprising porous silica microsphereshaving an average diameter of about 0.5 to about 35 μm, substantiallyall of said microspheres having a diameter ranging from about 0.5 toabout 1.5 times said average diameter; said microspheres consistingessentially of a plurality of substantially uniform-size colloidalparticles, having a silica surface, arranged in an interconnectedthree-dimensional lattice; said colloidal parpticles occupying less thanabout 50 volume percent of said microspheres with the remaining volumebeing occupied by interconnected pores having substantially uniform poresize distribution; said microspheres having a total concentration ofsilanol groups from about 8 to about 16 μmol/m², wherein themicrospheres are prepared according to a process comprising: contactingheat strengthened thermally-dehydroxylated porous silica microsphereshaving a surface concentration of silanol groups of less than about 5.5μmol/m² with water in the presence of HF or at least one basic activatorselected from the group consisting of quaternary ammonium hydroxides,ammonium hydroxide, and organic amines at a temperature of from aboutambient temperature to about 100° C. for sufficient time to generate thedesired concentration of silanol groups of from about 8 to about 16μmol/m².
 4. A chromatographic material as defined inclaimm 3, whereinthe heat strengthened thermally-dehydroxylated porous silicamicrospheres are contacted with water in the presence of at least onebasic activator selected from the group consisting of tetraalkylammoniumhydroxides, ammonium hydroxide, primary organic amines, and secondaryorganic amines.
 5. A chromatographic material as defined in claim 4,wherein the basic activator is added in a sufficient amount to generatea pH of from about 9 to about 10.5.
 6. A chromatographic material asdefined in claim 5, wherein the basic activator is selected from thegroup consisting of tetraalkylammonium hydroxides, ammonium hydroxide,and primary organic amines.
 7. A chromatographic material as defined inclaim 6, wherein the basic activator is a primary organic amine selectedfrom the group consisting of ethylenediamine, n-propylamine andn-butylamine.
 8. A chromatographic material as defined in claim 6,wherein free HF is present in a concentration of from about 50 to about400 ppm.
 9. A chromatographic material as defined in claim 3, whereinthe heat strengthened thermally-dehydroxylated porous silicamicrospheres are contacted with water in the presence of HF.
 10. Achromatographic material comprising improved porous silica microsphereshaving an average diameter of 0.5 to about 35 μm, substantially all ofsaid microspheres having a diameter ranging from about 0.5 to about 1.5times said average diameter; said microspheres consisting essentially ofa plurality of substantially uniform-size colloidal particles, having asilica surface, arranged in an interconnected three-dimensional lattice;said colloidal particles occupying less than about 50 volume percent ofsaid microspheres with the remaining volume being occupied byinterconnected pores having a substantially uniform pore sizedistribution; said microspheres having a completely silanized surface,wherein the microspheres are prepared according to a processcomprising:(a) contacting heat strengthened thermally-dehydroxylatedporous silica microspheres having surface concentration of silanolgroups of less than about 5.5 umol/m² with water in the presence of HFor at least one basic activator selected from the group consisting ofquaternary ammonium hydroxides, ammonium hydroxide, and organic aminesat a temperature of about ambient temperature to about 100° C. forsufficient time to generate a surface concentration of silanol groups offrom about 8 to about 16 μmol/m² ; and (b) contacting the porous silicamicrospheres prepared in step (a) with a silanizing agent at atemperature of from about 25° to about 100° C. for sufficient time togenerate a completely silanized surface.
 11. A chromatographic materialas defined in claim 10, wherein the microspheres have an averagediameter of from about 1.0 to about 10 μm.
 12. A chromatographicmaterial as defined in claim 10, wherein the heat strengthenedthermally-dehydroxylated porous silica microspheres are contacted withwater in the presence of at least one basic activator selected from thegroup consisting of tetraalkylammonium hydroxides, ammonium hydroxide,primary organic amines, and secondary organic amines.
 13. Achromatographic material as defined in claim 12, wherein the basicactivator is added in a sufficient amount to generate a pH of from about9 to about 10.5.
 14. A chromatographic material as defined in claim 13,wherein the basic activator is selected from the group consisting oftetraalkylammonium hydroxides, ammonium hydroxide, and primary organicamines.
 15. A chromatographic material as defined in claim 14, whereinthe basic activator is a primary organic amine selected from the groupconsisting of ethylenediamine, n-propylamine and n-butylamine.
 16. Achromatographic material as defined in claim 10, wherein the heatstrengthened thermally-dehydroxylated porous silica microspheres arecontacted with water in the presence of HF.
 17. A chromatographicmaterial as defined in claim 16, wherein free HF is present in aconcentration of from about 50 to about 400 ppm.